Method for fixation of biological tissues having mitigated propensity for post-implantation calcification and thrombosis and bioprosthetic devices prepared thereby

ABSTRACT

A method for fixation of biological tissues, and bioprosthetic devices prepared by such method. The method generally comprises the steps of A) fixing the tissue, B) treating the tissue with a mixture of i) a denaturant, ii) a surfactant and iii) a crosslinking agent, C) fabricating or forming the bioprosthesis (e.g., forming the tissue and attaching any non-biological components thereto) and D) subjecting the bioprosthesis to terminal sterilization.

FIELD OF THE INVENTION

The invention pertains generally to medical method/devices and moreparticularly to a method for fixing (e.g., tanning or crosslinking) andsterilizing biological tissue to i) decrease the fixed tissue'spropensity for post-implantation calcification and ii) decrease thethrombogenicity of the fixed tissue.

BACKGROUND OF THE INVENTION

Various types of implantable medical devices may be formed wholly orpartially of biological tissue that has been chemically “fixed” orpreserved. The technique used for chemical fixation of biologicaltissues typically requires exposure of the biological tissue to one ormore chemical agents that are capable of forming cross-linkages betweenconnective tissue protein molecules present in the tissue.

Examples of fixed biological tissues that have been used to formimplantable bioprostheses include cardiac valves, blood vessels, skin,dura mater, pericardium, ligaments and tendons. These biological tissuestypically contain connective tissue proteins (i.e., collagen andelastin) which act as the supportive framework of the tissue. Thepliability or rigidity of each biological tissue is largely determinedby the relative amounts of collagen and elastin present within thetissue and/or by the physical structure and confirmation of itsconnective tissue frame work.

Each Collagen molecule is made up of three (3) polypeptide chainsintertwined in a coiled helical confirmation. The Chemical fixatives(i.e., tanning agents) which are used to preserve biological tissuesgenerally form chemical cross-linkages between the polypeptide chainswithin a given collagen molecule (i.e., intramolecular crosslinkages),or between adjacent collagen molecules (i.e., intermolecularcrosslinkages).

Examples of chemical fixative agents which have been utilized tocross-link collagenous biological tissues include; aldehydes (e.g.,formaldehyde, glutaraldehyde, dialdehyde starch, para formaldehyde,glyceroaldehyde, glyoxal acetaldehyde, acrolein), diisocyanates (e.g.,hexamethylene diisocyanate), carbodiimides, photooxidabon, and certainpolyepoxy compounds (e.g., Denacol-810, −512, or related compounds). Ofthe various chemical fixatives available, glutaraldehyde is the mostwidely used. Glutaraldehyde is used as the fixative for manycommercially available bioprosthetic products, such as porcinebioprosthetic heart valves (i.e., the Carpentier-Edwards® stentedporcine bioprosthesis; Baxter Healthcare Corporation; Edwards CVSDivision, Irvine, Calif. 92714-5686), bovine pericardial heart valveprostheses (e.g., Carpentier-Edwards® Pericardial Bioprosthesis, BaxterHealthcare Corporation, Edwards CVS Division; Irvine, Calif. 92714-5686)and stentless porcine aortic prostheses (e.g., Edwards® PRIMA StentlessAortic Bioprosthesis, Baxter Edwards AG, Spierstrasse 5, GH6048, Horn,Switzerland).

One problem which has been associated with the implantation ofbioprosthetic materials is that the connective tissue proteins (i.e.,collagen and elastin) within these materials can become calcifiedfollowing implantation within the body. Such calcification can result inundesirable stiffening or degradation of the bioprosthesis. Two (2)types of calcification-intrinsic and extrinsic--are known to occur infixed collagenous bioprostheses, although the exact mechanism(s) bywhich such calcification occurs is unknown. Intrinsic calcification ischaracterized by the precipitation of calcium and phosphate ions withinthe fixed bioprosthetic tissue, including the collagen matrix andremnant cells. Extrinsic calcification is characterized by theprecipitation of calcium and phosphate ions within the thrombus,including adherent cells (e.g., platelets) to the bioprosthesis and thedevelopment of calcium phosphate-containing surface plaques on thebioprosthesis.

The factors that affect the rate at which fixed tissue bioprosthesesundergo calcification have not been fully elucidated. However, factorsthat are thought to influence the rate of calcification include:

a) patient's age;

b) existing metabolic disorders (i.e.,

c) hypercalcemia, diabetes, etc.);

d) dietary factors;

e) infection;

f) parenteral calcium administration;

g) dehydration;

h) distortion/mechanical factors;

i) inadequate coagulation therapy during initial period followingsurgical implantation; and

j) host tissue responses.

The factors that are thought to affect the propensity for platelets toadhere to a fixed bioprosthetic tissue include:

a) tissue damage;

b) diet;

c) surface properties of the tissue, including the nature of exposedcollagen (e.g., type I, IV, etc.);

d) metabolic changes;

e) coagulation;

f) hemodynamics

g) inflammation; and,

h) infection.

Various techniques have heretofore been proposed for mitigating the insitu calcification of glutaraldehyde-fixed bioprostheses. Included amongthese calcification mitigating techniques are the methods described inU.S. Pat. No. 4,885,005 (Nashef et al.) entitled Surfactant Treatment ofImplantable Biological Tissue To Inhibit Calcification; U.S. Pat. No.4,648,881 (Carpentier et al.) entitled Implantable Biological Tissue andProcess For Preparation Thereof; U.S. Pat. No. 4,976,733 (Girardot)entitled Prevention of Prosthesis Calcification; U.S. Pat. No. 4,120,649(Schechter) entitled Transplants; U.S. Pat. No. 5,002,2566 (Carpentier)entitled Calcification Mitigation of Bioprosthetic Implants; EP 103947A2(Pollock et al.) entitled Method For Inhibiting Mineralization ofNatural Tissue During Implantation and WO84/01879 (Nashef et al.)entitled Surfactant Treatment of Implantable Biological Tissue toInhibit Calcification; and, in Yi, D., Liu, W., Yang, J., Wang, B.,Dong, G., and Tan, H.; Study of Calcification Mechanism andAnti-calcification On Cardiac Bioprostheses Pgs. 17-22, Proceedings ofChinese Tissue Valve Conference, Beijing, China, June 1995.

There presently remains a need in the art for the development of newcalcification-mitigating methods for fixing (i.e., tanning andcrosslinking) and sterilizing biological tissues to providebioprosthetic devices which are a) less likely to become calcified andb) less thrombogenic, following implantation within a patient's body.

SUMMARY OF THE INVENTION

Broadly stated, the present invention provides a method for chemicalfixation and sterilization of biological tissue, comprising the stepsof:

1. HARVEST/PREPARATION OF TISSUE—harvesting and preparing a biologicaltissue.

2. FIXATION OF TISSUE—contacting the biological tissue with a fixativeagent such as an aldehyde (e.g., formaldehyde or glutaraldehyde) for afixation time period (e.g., when 0.625% glutaraldehyde is used as thefixative the time period will be 0.5 hours to 14 days) to effectcrosslinking of the connective tissue proteins within the tissue.

3. DSC TREATMENT—before or after performance of the fixation step (StepB above) the biological tissue is placed in contact with (e.g., immersedin) a denaturant/surfactant/crosslinking agent (DSC) solution at atemperature between 4 and 50 degrees C for a period of time ranging from90 minutes to 35 hours). This DSC treatment step is typically performedbefore transportation of the biological tissue into a clean room oraseptic environment, but may additionally or alternatively be performedat other times such as after assembly of the bioprosthesis as describedin more detail herebelow.

4. ASSEMBLY/FABRICATION OF PROSTHESIS—trimming or configuring thebiological tissue (if necessary) and attaching any requirednon-biological components (e.g., stents, frames, suture rings, otherhardware elements) thereto. This assembly/fabrication step is typicallyperformed in a clean room or aseptic environment;

5. TERMINAL STERILIZATION—subjecting the biological tissue to asterilant (e.g., a liquid sterilant such as 0.2-2.0% by weightglutaraldehyde solution) for a sterilization time period. A 0.625%glutaraldehyde solution may be used in combination with heat (i.e.warming above room temperature but below a temperature which would causedamage to the tissue of the bioprosthesis), as the sterilant.Alternatively, a suitable sterilant solution may comprise an osmoticallybalanced aqueous solution alone or in combination with a non-contactingsource of sterilization (e.g., radiation, electron beam, UV, or othersimilar expedient), or include an aqueous solution of glutaraldehyde incombination with the above-described DSC mixture or some components ofsuch DSC mixture. In instances where a 0.625% glutaraldehyde solution isused as the sterilant, the sterilization time period may be 1-6 days at37 degrees C or 1-2 days at 50 degrees C). This terminal sterilizationstep may be performed after packaging of the bioprosthesis in its finalcontainer, thereby eliminating the need for any subsequent handling ofthe bioprosthesis until the time of implantation.

Further in accordance with the invention, there are provided varioustypes of bioprosthetic articles that are wholly or partially formed oftissue that has been prepared by the above-summarizedfixation/sterilization method of the present invention. Examples ofspecific biological tissues which may be utilized to preparebioprosthetic articles in accordance with this invention include, butare not necessarily limited to: heart valves; venous valves; bloodvessels; ureter; tendon; dura mater; skin; pericardium; cartilage (e.g.,meniscus); ligament; bone; intestine (e.g., intestinal wall); andperiostium.

Further aspects and objects of the present invention will becomeapparent to those skilled in the relevant art, upon reading andunderstanding the detailed description of presently preferredembodiments set forth herebelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a general flow diagram of a calcification-mitigating treatmentmethod of the present invention;

FIG. 2 is a general flow diagram of a treatment method used as a controlin a study of the effectiveness of the present invention in mitigatingcalcification;

FIG. 3 is a general flow diagram of a treatment method used as a controlin a study of the effectiveness of the present invention in improvinghemocompatibility;

FIG. 4 is a scanning electron microscope image of the surface of tissuetreated in accordance with the method of FIG. 3; and

FIG. 5 is a scanning electron microscope image of the surface of tissuetreated in accordance with the method of FIG. 1.

Additional embodiments and aspects of the invention may become apparentto those of skill in the art upon reading and understanding of thedetailed description and specific examples set forth herebelow.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As shown in FIG. 1, the fixation/sterilization method of the presentinvention generally comprises six (6) steps as follows:

Step 1: Harvest/Prepare Biological Tissue

The desired biological tissue is harvested (i.e., surgically removed orcut away from its host animal). Thereafter, it is typically, trimmed orcut to size and washed with sterile water, balanced salt solution,saline or other suitable washing solution.

Step 2: Fixation of Biological Tissue

The biological tissue is then contacted with a crosslinking agent, suchas an aldehyde (e.g., formaldehyde, glutaraldehyde, dialdehyde starch),polyglycidyl either (e.g., Denacol 810), diisocyanates, photooxidation,or carbodiimide(s)) to crosslink the connective tissue proteins presentwithin the tissue. Due to the long standing use and experience withglutaraldehyde, a presently preferred fixative for use in this step is asolution of 0.2-2.0% by weight glutaraldehyde. For example, thebiological tissue may be immersed in a solution of 0.625% by weightglutaraldehyde buffered to a pH of approximately 7.4 by a suitablebuffer such as a phosphate buffer, for 0.5 hours to 14 days at 4-37degrees C.

Step 3: Treatment With Denaturant/Surfactant/Crosslinking Agent (DSCTreatment)

a. Before or after fixation of the tissue in Step 2, the tissue isimmersed in or otherwise contacted with a mixture containing i) acrosslinking agent, ii) a denaturing agent and iii) a surfactant (i.e.,a DSC solution). One preferred DSC solution is a mixture of i)formaldehyde, ii) ethanol and ii) surfactant (e.g., Tween 80™surfactant, available from ICI Americas, Brantford, Ontario). Apreferred formulation for the DSC solution is as follows:

Formaldehyde . . . 0.1-10.0% (more pref. 4±0.4%) by weight

Ethanol . . . 1% to less than 60% (more pref. 2.2±2.2%) by weight

Tween 80 . . . 0.1-5.0% (more pref. 1.2 ±0.12%) by weight

The tissue is preferably immersed in the DSC solution for 2 to 24 hoursand typically about 9 hours. During this immersion period, the DSCsolution is maintained at a temperature of 4-50 degrees C, and typicallyabout 20-37 degrees C.

Those skilled in the art will appreciate that various alternativechemical compounds or solutions may be substituted for each component ofthe DSC solution, as follows:

Potential Alternative Denaturing Agents

A. Alcohols/Solvents: e.g., ethanol, isopropyl alcohol, acetone, ethersof small alkyl size (methyl, ethyl, propyl, isopropyl)

B. Acidified Ethers: e.g., sulfuric acid/ether mixture

C. Ketones: e.g., methyl ethyl ketone (MEK)

D. Chlorofluorocarbon Solvents: e.g., Genesolve™ (Allied Signal, Inc.,Morristown, N.J.)

E. Glycols: glycerol ethylene glycol, polyethylene glycols of varyingmolecular weight

F. Chaotropic Agents: e.g., urea, guanidine hydrochloride, guanidinethiocyanate potassium iodide

G. High Concentration Salt Solutions: e.g., lithium chloride, sodiumchloride, cesium chloride.

Potential Alternative Surfactants**

** these surfactant compounds can be used individually or in mixturessuch as deoxycholate/Triton or commercially-available mixtures such asMicro-80/90.

A. Anionic Surfactants: e.g., esters of lauric acid, including but notlimited to sodium laurel sulfate (also called sodium dodecyl sulfate)

B. Alkyl sulfonic acid salts: e.g., 1-decanesulfonic acid sodium salt

C. Non-ionic compounds: e.g., compounds based on the polyoxyethyleneether structures (including Triton X-100, 114, 405, N-101 availablecommercially from Sigma Chemical, St. Louis, Mo.), and relatedstructures; Pluronic and Tetronic surfactants (available commerciallyfrom BASF Chemicals, Mount Olive, N.J.)

D. Alkylated Phenoxypolyethoxy Alcohols: e.g., NP40, Nonidet P40,Igepal, CA630, hydrolyzed/functionalized animal and plant compoundsincluding, Tween 80, Tween 20, octyl-derivatives, octyl b-glucoside,octyl b-thioglucopyranoside, deoxycholate and derivatives thereof,zwitterionic compounds, 3-([cholamidopropyl]-dimethylammonio)-1-propanesulfonate (CHAPS), 3-([cholamidopropyl]-dimethylammonio)-2-hydroxy-1-propanesulfonate (CHAPSO) available from PierceBiotec Company, Rockford, Ill.

Potential Alternative Crosslinking Agents

A. Aldehydes: formaldehyde, glutaraldehyde, paraformaldehyde,glyceraldehyde, glyoxal acetaldehyde or acrolein, dialdehyde starch

B. Epoxides: e.g., any of the various Denacols and their individualreactive species, including mono, di, tri, and multi-functionalizedepoxides

C. Carbodiimides

D. Diisocyanates

E. Photooxidation

Step 4: Assembly/Fabrication of Prosthesis

After completion of Steps 1-3, and irrespective of the order in whichSteps 2 and 3 were performed, the tissue is rinsed with a suitablerinsing solution such as isotonic saline or 0.625% glutaraldehyde.Thereafter, the tissue may be transported into a clean room or asepticenvironment, and may be further trimmed or shaped (if necessary) andattached to or assembled with any non-biological components (e.g.,stents, frames, suture rings, conduits, segments of polyester mesh toprevent suture tear-through, etc . . . ) To form the desired implantablebioprosthetic device. Examples of such assembled bioprosthetic devicesinclude porcine bioprosthetic heart valves (i.e., theCarpentier-Edwards® stented porcine bioprosthesis; Baxter HealthcareCorporation; Edwards CVS Division, Irvine, Calif. 92714-5686), bovinepericardial heart valve prostheses (e.g., Carpentier-Edwards®Pericardial Bioprosthesis, Baxter Healthcare Corporation, Edwards CVSDivision; Irvine, Calif. 92714-5686), stentless porcine aorticprostheses (e.g., Edwards® PRIMA Stentless Aortic Bioprosthesis, BaxterEdwards AG, Spierstrasse 5, GH6048, Horn, Switzerland), andbio-mechanical ventricular assist devices (e.g., the Novacor N-100PCmodel; Novacor, Oakland, Calif.).

Step 5: Treatment With Denaturant/Surfactant/Crosslinking Agent (DSCTreatment) (Optional)

Optionally, the DSC treatment described in Step 3 above may be carriedout at this point in the procedure instead of as the third (3rd) step ofthe procedure. Or, if such DSC treatment has already been performed asthe third (3rd) step of the procedure, it may be repeated at this pointin the procedure (e.g., as the fifth (5th) step of the procedure).

Step 6: Terminal Sterilization

The bioprosthesis is immersed in or contacted with a terminal sterilantand heated for a period of time sufficient to ensure sterility of thebioprosthesis until the time of implantation. This terminalsterilization procedure is preferably carried out in the sealedcontainer or package in which the bioprosthesis will be shipped andstored until the time of implantation.

A preferred terminal sterilant is 0.2-2.0% by weight glutaraldehyde, andmost preferably about 0.25% by weight glutaraldehyde. Although DSCsolution or compounds of the DSC solution can also be used. Thepreferred terminal sterilization time and temperature is 1-6 days at 37°C. or 1-2 days at 50° C.

Preparation of a Stented Pericardial Valve Bioprosthesis

The following is an example of the manner in which a stented pericardialbioprosthetic heart valve may be manufactured in accordance with themethod of the present invention.

STEP 1: A bovine pericardial sac is obtained from a slaughterhouse,placed on ice, and transported to the location at which thebioprosthesis will be manufactured. Thereafter, the tissue is defattedand trimmed.

STEP 2: The tissue is washed with sterile isotonic saline solution andis thereafter immersed in a solution of 0.625% by weight glutaraldehydebuffered to a pH of approximately 7.4 by a suitable buffer such as aphosphate buffer, for approximately thirty minutes at room temperature.This results in crosslinking of the collagen present within the tissue.

STEP 3: The tissue is then removed from the fixative solution used inStep 2 and rinsed thoroughly with an aqueous solution of 0.625% (byweight) glutaraldehyde. Sometime thereafter, the DSC Treatment of Step 3is carried out by immersing the tissue in DSC solution for 2 hours atambient temperature. The DSC solution has the following formula:

Formaldehyde . . . 4.0±0.4% by weight

Ethanol . . . 22±2.2% by weight

Tween 80 . . . 1.2±0.12% by weight

STEP 4: After completion of the DSC Treatment of Step 3, the tissue isremoved from the DSC solution and leaflets are formed. Thereafter, theleaflets are mounted upon and sutured to a stent. Also, aneedle-penetrable suture ring is attached about the inflow end of thevalve to facilitate suturing of the bioprosthesis to the native tissueof the host patient. This completes the assembly and fabrication of thebioprosthetic heart valve.

STEP 5: Subject the finished valve after inspection to DSC treatmentagain for 9 hours at 37° C.

STEP 6: After the bioprosthesis is removed from the DSC solution it istransferred to a container which has been pre-filled with 0.25%glutaraldehyde aqueous solution buffered to a pH of 7.4 with sodiumhydroxide such that the bioprosthetic valve is fully immersed in thebuffered glutaraldehyde solution. Thereafter, the container is sealedand placed in an oven where it is heated to a terminal sterilizationtemperature of 37.5±2.5 degrees C for 25-27 hours. Thereafter, thecontainer is cooled to room temperature and shipped to the hospital orother location(s) where it is stored until the time of implantation ofthe bioprosthetic valve.

Studies were performed to objectively assess the benefits of theabove-described process in mitigating calcification and improvinghemocompatibility. First, the new process was compared with a process asshown in FIG. 2, which includes a terminal sterilization step after“assembly,” but does not include any intermediate DSC steps. Theassembly step in this case consisted of cutting pieces of fixed tissueinto circles for subcutaneous implantation in animals. After a suitableperiod, the tissues were explanted and analyzed for calcium content, anindicator of calcification rate. In a second study, the new process wascompared with a process as shown in FIG. 3, which includes intermediateDSC steps but does not include a terminal sterilization step. Theresulting tissues were then exposed to whole blood for a period and thesurfaces photographed using a scanning electron microscope to visiblydetermine any differences.

In the first comparison study, control tissues were generated bytraditional glutaraldehyde fixation in conjunction with a terminalsterilization step as described above. In these tests, circular piecesof tissue prepared by the new method and by the control method weresurgically placed in either the subcutaneous space in rats and rabbits,or in a pocket created in the paravertebral muscle in the rabbit. Afterthirty days implantation the tissues were excised from the host tissue,rinsed, and calcium content is determined by atomic absorptionspectroscopy. The study results are as follows:

Determination of Calcium Content by Atomic Absorption SpectroscopyTissues surgically implanted for Thirty days (ug Ca/mg tissue, dryweight)

Implant site: n: Control: n: New process: Rat subcutaneous 6 124.04 ±19.69 6 0.07 ± 0.18 Rabbit subcutaneous 6  95.35 ± 48.69 6 9.07 ± 7.88Rabbit intramuscular 5 165.13 ± 22.88 4 86.15 ± 51.58

As is apparent, tissue produced according to the new process demonstratereduced calcification in rat and rabbit implantation assays, anddemonstrate the superiority of the new tissue preparation methodcompared to the control method.

The second study shows that tissues prepared according to the new methodalso demonstrate reduced adhesion of blood cells compared to atraditional treatment methods. The control method for the second studyis seen in FIG. 3, and includes traditional glutaraldehyde fixation inconjunction with intermediate DSC steps. Scanning electron microscopywas used to assess the degree of adhesion of blood cells after exposureof anticoagulated whole human blood to tissues prepared according to thenew method and to tissues prepared by the control method of FIG. 3.After a one hour exposure to whole human blood, the tissues were rinsed,critical point dried, lightly coated with gold and imaged in the SEImode at an accelerating voltage of 5 kV in a JEOL 6300F field emissionscanning electron microscope. The results are shown in FIGS. 4 and 5.

FIG. 4 shows an image from a representative area on the control tissuesafter exposure to whole human blood in vitro. Note numerous adherentcells and platelets on the surface. FIG. 5 shows an image from arepresentative area on tissues prepared by the new process afterexposure to whole human blood in vitro. Note the nearly complete absenceof adherent cells and platelets on the surface. These images demonstratethe improved hemocompatibility (i.e., reduced thrombogenicity) of thematerial prepared using the new process compared to the control method.

The invention has been described hereabove with reference to certainpresently preferred embodiments or examples only, and no effort has beenmade to exhaustively describe all possible embodiments or examples ofthe invention. Those skilled in the art will recognize that variousmodifications, additions and changes may be made to the particularembodiments and examples described hereabove without departing from theintended spirit and scope of the invention. Accordingly, it is intendedthat all such modifications, additions and changes be included withinthe scope of the following claims.

What is claimed is:
 1. A method for preparation of a bioprosthesis thatcontains connective tissue protein, said method comprising the steps of:a. contacting the connective tissue protein within the biological tissuewith a fixative agent so as to cause crosslinking of connective tissueprotein; b. contacting the biological tissue with a mixture of adenaturant, a surfactant and a crosslinking agent; c. assembling thebioprosthesis by performing any required shaping of the biologicaltissue and attaching any non-biological components of the prosthesis tothe biological tissue; and, d. after completion of steps a. b and c.immersing the bioprosthesis in a liquid terminal sterilization solutionand heating said terminal sterilization solution to a temperaturebetween 35-50 degrees C.
 2. The method of claim 1 wherein the fixativeagent used in Step A comprises an aldehyde.
 3. The method of claim 1wherein the fixative agent used in Step A is selected from the groupconsisting of: a. diisocyanates, b. carbodiimides, c. photooxidation;and d. polyepoxy compounds.
 4. The method of claim 1 wherein Step Acomprises immersing the biological tissue in a solution containing0.2-2.0% by weight glutaraldehyde for 0.5 hours-14 days at 4-37 degreesC.
 5. The method of claim 1 wherein Step A is performed prior to Step B.6. The method of claim 1 wherein Step B is performed before Step A. 7.The method of claim 1 wherein Step B is performed after Step C.
 8. Themethod of claim 1 wherein Step B is performed before Step A and againbefore Step C.
 9. The method of claim 1 wherein Step B is performedbefore Step A and again after Step C.
 10. The method of claim I whereinStep B is performed after Step A and again after Step C.
 11. The methodof claim I wherein Step B is performed before Step A, again after Step Aand again after Step C.
 12. The method of claim 1 wherein thecrosslinking agent of the mixture used in Step B is an aldehyde.
 13. Themethod of claim 1 wherein the denaturing agent of the mixture used inStep B is an alcohol.
 14. The method of claim 1 wherein the denaturingagent of the mixture used in Step B is selected from a group consistingof. a. acidified ethers; b. ketones; c. chlorofluorocarbon solvents; d.glycols; e. chaotropic agents; and f. high concentration salt solutions.15. The method of claim 1 wherein the surfactant agent of the mixtureused in Step B is selected from a group consisting of: a. anionicsurfactants; b. alkyl sulfonic acid salts; c. non-ionic compounds; andd. alkylated phenoxypolyethoxy alcohols.
 16. The method of claim 1wherein the mixture used in Step B is a DSC solution which comprises: a.Formaldehyde . . . 0.1-10.0% by weight; b. Ethanol . . . 1% to less than60% by weight; and, c. Tween 80 . . . 0.1-5.0% by weight.
 17. The methodof claim 16 wherein the mixture used in Step B is maintained at atemperature of at least 4 degrees C. but below 50 degrees C. while incontact with the biological tissue.
 18. The method of claim 1 whereinthe DSC solution used in Step B is maintained at a temperature of atleast 4 degrees C. but below 50 degrees C. while in contact with thebiological tissue.
 19. The method of claim 1 wherein Step D furthercomprises: maintaining the temperature of said liquid terminalsterilization solution between 35-50 degrees C. for a period of timesufficient to ensure the sterility of the bioprosthesis until the timeof implantation.
 20. The method of claim 1 wherein the sterilant used inStep D is selected from the group of sterilants consisting of: a. liquidsterilants; and, b. a non-contacting sterilization source.
 21. Themethod of claim 1 wherein Step D comprises immersing the bioprosthesisin a liquid terminal sterilization solution and heating said terminalsterilization solution to a temperature between 37-50 for a period oftime sufficient to ensure the sterility of the bioprosthesis until thetime of implantation.
 22. The method of claim 21 wherein Step D iscarried out in a sealed container and further comprises allowing thebioprosthesis to remain within said sealed container until the time ofimplantation.
 23. The method of claim 21 wherein said terminalsterilization solution comprises an aqueous solution of 0.2-2.0% byweight glutaraldehyde buffered to a pH of approximately 7.4.
 24. Themethod of claim 21 wherein said terminal sterilization solutioncomprises an osmotically balanced salt solution in the absence of otherchemical sterilants and wherein the solution is heated to a temperatureof at least 45 degrees C.
 25. The method of claim 21 wherein theterminal sterilization solution comprises osmotically balanced saltsolution in combination with at least one chemical sterilant.
 26. Themethod of claim 21 wherein the terminal sterilization solution comprisesat least one component selected from i) a denaturant, ii) a surfactantand iii) a crosslinking agent.
 27. The method of claim 1 wherein Step Dcomprises: a. dispensing into a container a quantity of a terminalsterilant solution comprising 0.2-2.0% by weight glutaraldehyde bufferedto a pH of approximately 7.4; b. immersing the bioprosthesis in saidterminal sterilant solution within said container; c. sealing saidcontainer; d. heating said container, and the terminal sterilantsolution and bioprosthesis contained therein, to a temperature of 37-50degrees C. for 1-6 days; e. cooling said container, and the terminalsterilant solution and bioprosthesis contained therein, to roomtemperature; and, f. allowing said container to remain sealed until itis desired to implant the bioprosthesis in a mammalian patient.
 28. Abioprosthesis comprising biological tissue having reduced potential forpost-implantation calcification and thrombogenicity which has been fixedand sterilized by a method which comprises the steps of: a. crosslinkingthe connective tissue protein within the biological tissue with afixative agent: b. contacting the biological tissue with a mixture of adenaturant, a surfactant and a crosslinking agent; c. assembling thebioprosthesis by performing any required shaping of the biologicaltissue and attaching any non-biological components of the prosthesis tothe biological tissue; and, d. after completion of steps a, b and c,immersing the bioprosthesis in a liquid terminal sterilization solutionand heating said terminal sterilization solution to a temperaturebetween 35-50 degrees C.
 29. The bioprosthesis of claim 28 wherein thefixative agent used in Step A comprises an aldehyde.
 30. Thebioprosthesis of claim 28 wherein the aldehyde fixative agent used inStep A is selected from the group consisting of: a. diisocyanates, b.carbodiimides, c. photooxidation; and d. polyepoxy compounds.
 31. Thebioprosthesis of claim 28 wherein Step A comprises immersing thebiological tissue in a solution containing 0.2-2.0% by weightglutaraldehyde for 0.5 hours-14 days at 4-37 degrees C.
 32. Thebioprosthesis of claim 28 wherein Step A is performed prior to Step B.33. The bioprosthesis of claim 28 wherein Step B is performed beforeStep A.
 34. The bioprosthesis of claim 28 wherein Step B is performedafter Step C.
 35. The bioprosthesis of claim 28 wherein Step B isperformed before Step A and before Step C.
 36. The bioprosthesis ofclaim 28 wherein Step B is performed before Step A and again after StepC.
 37. The bioprosthesis of claim 28 wherein Step B is performed afterStep A and again after Step C.
 38. The bioprosthesis of claim 28 whereinStep B is performed before Step A, again after Step A and again afterStep C.
 39. The bioprosthesis of claim 28 wherein the crosslinking agentof the mixture used in Step B is an aldehyde.
 40. The bioprosthesis ofclaim 28 wherein the denaturing agent of the mixture used in Step B isan alcohol.
 41. The bioprosthesis of claim 28 wherein the denaturingagent of the mixture used in Step B is selected from a group consistingof: a. acidified ethers; b. ketones; c. chlorofluorocarbon solvents; d.glycols; e. chaotropic agents; and f. high concentration salt solutions.42. The bioprosthesis of claim 28 wherein the surfactant agent of themixture used in Step B is selected from a group consisting of: a.anionic surfactants; b. alkyl sulfonic acid salts; c. non-ioniccompounds; and d. alkylated phenoxypolyethoxy alcohols.
 43. Thebioprosthesis of claim 28 wherein the mixture used in Step B is a DSCsolution which comprises: a. Formaldehyde . . . 0.1-10.0% by weight; b.Ethanol . . . 1% to less than 60% by weight; and, c. Tween 80 . . .0.1-5.0% by weight.
 44. The bioprosthesis of claim 43 wherein themixture used in Step B is maintained at a temperature of at least 4degrees C but below 50 degrees C while in contact with the biologicaltissue.
 45. The bioprosthesis of claim 28 wherein the mixture used inStep B is maintained at a temperature of at least 4 degrees C. but below50 degrees C while in contact with the biological tissue.
 46. Thebioprosthesis of claim 28 wherein Step B comprises: a. immersing thebiological tissue in a solution containing 0.1-10.0% by weightformaldehyde, 1% to less than 60% by weight ethanol and 0.1-5.0% byweight of a surfactant; and, b. maintaining said solution at atemperature of at least 4 degrees C. but below 50 degrees C. for 1-36hours.
 47. The bioprosthesis of claim 28 wherein the sterilant used inStep D is selected from the group of sterilants consisting of: a. liquidsterilants; and, b. a non-contacting sterilization source.
 48. Thebioprosthesis of claim 28 wherein Step D of the method by which thebioprosthesis is fixed and sterilized further comprises: maintaining thetemperature of said liquid terminal sterilization solution between 35-50degrees C. for a period of time sufficient to ensure the sterility ofthe bioprosthesis until the time of implantation.
 49. The bioprosthesisof claim 48 wherein Step D is carried out in a sealed container andfurther comprises allowing the bioprosthesis to remain within saidsealed container until the time of implantation.
 50. The bioprosthesisof claim 48 wherein said terminal sterilization solution comprises anaqueous solution of 0.2-2.0% by weight glutaraldehyde buffered to a pHof approximately 7.4.
 51. The bioprosthesis of claim 48 wherein saidterminal sterilization solution comprises an osmotically balanced saltsolution in the absence of other chemical sterilants and wherein thesolution is heated to a temperature of at least 45 degrees C.
 52. Thebioprosthesis of claim 48 wherein the terminal sterilization solutioncomprises osmotically balanced salt solution in combination with atleast one chemical sterilant.
 53. The bioprosthesis of claim 48 whereinthe terminal sterilization solution comprises at least one componentselected from i) a denaturant, ii) a surfactant and iii) a crosslinkingagent.
 54. The bioprosthesis of claim 28 wherein Step D comprises: a.dispensing into a container a quantity of a terminal sterilant solutioncomprising 0.2-2.0% by weight glutaraldehyde buffered to a pH ofapproximately 7.4; b. immersing the bioprosthesis in said terminalsterilant solution within said container; c. sealing said container; d.heating said container, and the terminal sterilant solution andbioprosthesis contained therein, to a temperature of 37-50 degrees C.for 1-6 days; e. cooling said container, and the terminal sterilantsolution and bioprosthesis contained therein, to room temperature; and,f. allowing said container to remain sealed until it is desired toimplant the bioprosthesis in a mammalian patient.
 55. A method accordingto claim 1 wherein Step b is performed prior to Step c, and wherein Stepb is then repeated between Steps c and d.
 56. A bioprosthesis accordingto claim 1 wherein Step b of the method by which the bioprosthesis isfixed and sterilized is performed prior to Step c, and wherein Step b isthen repeated between Steps c and d.